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The amiloride-sensitive epithelial Na ؉ channel (ENaC) plays a critical role in the maintenance of alveolar fluid balance. It is generally accepted that reactive oxygen and nitrogen species can inhibit ENaC activity and aggravate acute lung injury; however, the molecular mechanism for free radical-mediated ENaC inhibition is unclear. Previously, we showed that the expression of the ␣-subunit of ENaC, ␣-ENaC, which is indispensable for ENaC activity, is repressed by Ras activation in salivary epithelial cells. Here, we investigated whether exogenous H 2 O 2 modulates ␣-ENaC gene expression in lung epithelial cells through a similar molecular mechanism. Utilizing transient transfection reporter assays and sitedirected mutagenesis analyses, we found that the glucocorticoid response element (GRE), located at ؊1334 to ؊1306 base pairs of the ␣-ENaC 5-flanking region, is the major enhancer for the stimulated ␣-ENaC expression in A549 lung epithelial cells. We further demonstrate that the presence of an intact GRE is necessary and sufficient for oxidants to repress ␣-ENaC expression. Consistent with our hypothesis, exogenous H 2 O 2 -mediated repression of ␣-ENaC GRE activity is partially blocked by either a specific inhibitor for extracellular signalregulated kinase (ERK) pathway activation, U0126, or dominant negative ERK, suggesting that, in part, activated ERK may mediate the repressive effects of H 2 O 2 on ␣-ENaC expression. In addition, overexpression of thioredoxin restored glucocorticoid receptor action on the ␣-ENaC GRE in the presence of exogenous H 2 O 2 . Taken together, we hypothesize that oxidative stress impairs Na ؉ transport activity by inhibiting dexamethasone-dependent ␣-ENaC GRE activation via both ERKdependent and thioredoxin-sensitive pathways. These results suggest a putative mechanism whereby cellular redox potentials modulate the glucocorticoid receptor/ dexamethasone effect on ␣-ENaC expression in lung and other tight epithelia.

The functional expression of the amiloride-sensitive epithelial sodium channel (ENaC) in select epithelia is critical for maintaining electrolyte and fluid homeostasis. Although ENaC activity is strictly dependent upon its ␣-subunit expression, little is known about the molecular mechanisms by which cells modulate ␣-ENaC gene expression. Previously, we have shown that salivary ␣-ENaC expression is transcriptionally repressed by the activation of Raf/extracellular signal-regulated protein kinase pathway. Here, this work further investigates the molecular mechanism(s) by which ␣-ENaC expression is regulated in salivary epithelial Pa-4 cells. A region located between ؊1.5 and ؊1.0 kilobase pairs of the ␣-ENaC 5-flanking region is demonstrated to be indispensable for the maximal and Ras-repressible reporter expression. Deletional analyses using heterologous promoter constructs reveal that a DNA sequence between ؊1355 and ؊1269 base pairs functions as an enhancer conferring the high level of expression on reporter constructs, and this induction effect is inhibited by Ras pathway activation. Mutational analyses indicate that full induction and Ras-mediated repression require a glucocorticoid response element (GRE) located between ؊1323 and ؊1309 base pairs. The identified ␣-ENaC GRE encompassing sequence (؊1334/؊1306) is sufficient to confer glucocorticoid receptor/dexamethasonedependent and Ras-repressible expression on both heterologous and homologous promoters. This report demonstrates for the first time that the cross-talk between glucocorticoid receptor and Ras/extracellular signal-regulated protein kinase signaling pathways results in an antagonistic effect at the transcriptional level to modulate ␣-ENaC expression through the identified GRE. In summary, this study presents a mechanism by which ␣-ENaC expression is regulated in salivary epithelial cells.

Previously, we have demonstrated that oxidative stress or Ras/ERK activation leads to the transcriptional repression of ␣-subunit of epithelial Na ؉ channel (ENaC) in lung and salivary epithelial cells. Here, we further investigated the coordinated molecular mechanisms by which ␣-ENaC expression is regulated. Using both stable and transient transfection assays, we demonstrate that the overexpression of high mobility group protein I-C (HMGI-C), a Ras/ERK-inducible HMG-I family member, represses glucocorticoid receptor (GR)/dexamethasone (Dex)-stimulated ␣-ENaC/reporter activity in salivary epithelial cells. Northern analyses further confirm that the expression of endogenous ␣-ENaC gene in salivary Pa-4 cells is suppressed by an ectopic HMGI-C overexpression. Through yeast two-hybrid screening and co-immunoprecipitation assays from eukaryotic cells, we also discovered the interaction between HMGI-C and PIAS3 (protein inhibitor of activated STAT3 (signal transducer and activator of transcription 3)). A low level of ectopically expressed PIAS3 cooperatively inhibits GR/Dex-dependent ␣-ENaC transcription in the presence of HMGI-C. Reciprocally, HMGI-C expression also coordinately enhances PIAS3-mediated repression of STAT3-dependent transactivation. Moreover, overexpression of antisense HMGI-C construct is capable of reversing the repression mediated by Ras V12 on GR-and STAT3-dependent transcriptional activation. Together, our results demonstrate that Ras/ERKmediated induction of HMGI-C is required to effectively repress GR/Dex-stimulated transcription of ␣-ENaC gene and STAT3-mediated transactivation. These findings delineate a network of inhibitory signaling pathways that converge on HMGI-C⅐PIAS3 complex, causally associating Ras/ERK activation with the repression of both GR and STAT3 signaling pathways in salivary epithelial cells.

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